Study of skin inflammation

The project aims to investigate the anti-inflammatory effect on the skin of extracts of plant origin and pure molecules on a non-tumor cell line of human keratinocytes. Keratinocytes are the main cells of the epidermis and have the specific task of producing keratin, a fibrous protein capable of protecting the epidermis from external insults. These cells are involved in numerous inflammatory skin diseases such as, for example, psoriasis and atopic dermatitis. Furthermore, keratinocytes secrete, in response to pro-inflammatory stimuli, metalloprotease-9 (MMP-9), a Ca-Zn dependent protease whose main function is the regulation of the extracellular matrix. This enzyme is in fact capable of degrading gelatin, type IV collagen, fibronectin, elastin and numerous other protein substrates. As a consequence of the degradation of basement membrane components by MMP-9, cells of the immune system, such as macrophages, neutrophils and T lymphocytes, reach the epidermis during inflammatory skin diseases.

MMP-9 is associated with numerous inflammatory processes, such as autoimmune diseases and psoriasis, and is now considered an enzyme closely involved in skin diseases. MMP-9 is induced in the keratinocyte in response to pro-inflammatory stimuli, such as those produced by cytokines such as TNF-α, IL-1β and IL-6. Recently, it has been demonstrated that histamine, one of the main chemical mediators of inflammation, induces an increase in MMP-9 expression in human keratinocytes. This process could represent one of the mechanisms through which these cells contribute to skin pathologies. Keratinocytes respond promptly to stimuli deriving from lymphocytic inflammation. Psoriasis is associated with Crohn’s disease with which it shares many inflammatory aspects: in the skin, macrophages and T lymphocytes produce high levels of TNF-α, IFN-γ and IL-17A. These cytokines induce the production of proliferative factors (VEGF, IL-8) and chemokines by keratinocytes, which hyperproliferate and amplify inflammation.

In atopic dermatitis, on the other hand, keratinocytes participate in allergic inflammation. Alongside the typical cytokines of autoimmune inflammation (TNF-α, IFN-γ and IL-17A), high levels of IL-4 produced by lymphocytes following skin sensitization prevail in atopic dermatitis. Keratinocytes respond to IL-4 stimulation by altering their differentiation state and skin barrier, but also by producing pro-allergic mediators (TSLP, IL-6, eotaxins, MMPs) which attract eosinophilic and basophilic granulocytes, and activate mast cells to the production of histamine. In atopic dermatitis the characteristic signs are edema, pruritus and skin lesion.

Inhibition of TNF-α and IL-17A (infliximab and secukinumab) are therapeutic options that lead to a marked improvement in psoriasis. The inhibition of IL-4, on the other hand, has given important therapeutic results in atopic dermatitis.

Methods used in the laboratory

In vitro assays

The assays involve the use of HaCaT cells (human keratinocytes) treated with various pro-inflammatory stimuli known to be involved in inflammatory skin pathologies.

  1. Measurement of NF-κB-driven transcription following pro-inflammatory stimulation with TNF-α, IL-6, histamine, bacterial LPS, and UV light.
  2. Measurement of NF-κB translocation following pro-inflammatory stimulation with TNF-α, IL-6, histamine, bacterial LPS and UV light.
  3. Measurement of translocation of NRF2.
  4. Secretion, gene expression (promoter activity and measurement of mRNA levels) and catalytic activity of metalloproteases following pro-inflammatory and pro-oxidant stimuli typical of skin pathologies.
  5. Effect on the release of pro-inflammatory cytokines and chemokines (IL-6, IL-8, eotaxins, adhesion molecules) and on the release of VEGF.
  6. Evaluation of markers of skin differentiation (keratin 1 and 10, involucrin)
  7. Measurement, by real time RT-PCR, of typically pro-inflammatory genes at the skin level (e.g. iNOS, COX-2);
  8. Skin permeability studies, using Franz cells.